Our research analyzes molecular mechanisms regulating chorion (eggshell) gene expression in time and space within ovarian follicle cells during Drosophila oogenesis. The specific amplification of the two major chorion gene clusters remains a primary focus of study. We will further delineate cis-regulatory sequences required for amplification by analyzing control regions mutated in vitro via P element-mediated transformation. The role of transcriptional control elements, including enhancers, in defining the tissue-specificity of amplification will be tested. This will require further definition of the cis-regulatory elements of the s15, s18 and possibly other chorion genes. Replication origins used during amplification will be mapped to determine if they correspond to genetically defined essential sequences for amplification. We will attempt to develop a general method for mapping Drosophila replication origins using minichromosomes which can replicate extrachromosomally in Drosophila cells. We will analyze and clone unlinked genes which are required for chorion gene amplification, including fs(3)293 and fs(3)272. Additional genes in this class will be identified using a method of insertional mutagenesis by single P element hopping which greatly simplifies the task of cloning genetically defined Drosophila genes. Follicle cell nuclear proteins binding to amplification control regions will be purified and used to identify the corresponding genes.